1. Field of the Invention
The invention relates to synthetic oligonucleotides and to their use in molecular biology applications and in the antisense therapeutic approach.
2. Summary of the Related Art
Oligonucleotides have become indispensible tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Carruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager el al., Biochemistry 27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
The routine synthesis of oligonucleotides is presently carried out using various N-acyl protecting groups for the nucleoside bases, such as isobutyryl (for guanine), and benzoyl for adenine and cytosine. After the synthesis of the oligonucleotides is carried out using either phosphoramidite chemistry or H-phosphonate chemistry, the protecting groups are removed by treatment with ammonia at 55-60.degree. C. for 5-10 hours. Using these protecting groups, PO oligonucleotides and other modified oligonucleotides can be synthesized. But in certain instances where modified oligonucleotides are functionalized with base-sensitive groups, the functionalities often get removed while the deprotection is being carried out.
This limitation in the oligonucleotide synthesis procedure has resulted in the inability to synthesize certain modified oligonucleotides that may have considerable utility. For example, oligonucleotides containing methyl phosphotriester internucleotide linkages could have many beneficial properties, because the methyl phosphotriester group is nonionic, but is similar in size and molecular shape to the phosphodiester linkage. Such nonionic methyl phosphotriester linkages could result in a reduction in oligonucleotide side effects that are attributable to the polyanionic character of the oligonucleotides. For example, Galbraith et al., Antisense Research and Development 4: 201-206 (1994) disclose complement activation by oligonucleotides. Henry et al., Pharm. Res. 11: PPDM8082 (1994) discloses that oligonucleotides may potentially interfere with blood clotting.
The art has recognized the desirability of incorporating methyl phosphotriester internucleotide linkages into oligonucleotides and many attempts have been made to make and use such oligonucleotides. However, these attempts have subsequently been discovered to be unsuccessful. Miller et al., J. Am. Chem. Soc. 93: 6657-6665 (1971), discloses alleged methylphosphotriester DNA synthesis by methylation of the phosphate using p-toluenesulphonyl chloride and methanol. Moody et al., Nucl. Acids Res. 17: 4769-4783 (1989), discloses regiospecific inhibition of DNA duplication by oligonucleotides synthesized according to the method of Miller et al.. Buck et al., Science 248: 208-212 (1990), discloses that oligonucleotides according to Moody et al. inhibit viral infectivity of HIV-1. However, Buck et al., Science 249: 125-126 (1990), retracts the earlier Buck et al. report and discloses that oligonucleotides synthesized according to this method do not contain methyl phosphotriester internucleotide linkages.
The difficulty in synthesizing oligonucleotides having methyl phosphotriester internucleotide linkages is due to the lability of the methyl ester bond under the oligonucleotide synthesis conditions used in the steps of deprotecting the nucleoside bases and cleaving the oligonucleotides from the solid support. Alul et al., Nucl. Acids Res. 19: 1527-1532 (1991), addressed the problem of cleaving the oligonucleotide from the solid support by introducing an oxalyl-type linker that can be cleaved under conditions that preserve the methyl ester bond. However, the problem of base deprotection was not addressed, so they were only able to synthesize methyl phosphotriester-linked thymidines, which lack an exocyclic amino group and thus do not require deprotection. Kuijpers et al., Nucl. Acids Res. 18: 5197-5205 (1990), attempted to address the deprotection problem by treating the nucleoside bases for 43 hours in potassium carbonate/methanol. Unfortunately, NMR analysis of their oligonucleotides revealed that considerable demethylation had occurred, resulting oligonucleotides having a mixture of methylphosphotriester and phosphodiester linkages. Similarly, Vinogradov et al., Tetrahedron Lett. 34: 5899-5902 (1993), attempted to solve the problem by using an isopropoxyacetyl group to protect the nucleoside bases, but found that at least 35-40% demethylation still occurred. Most recently, Hayakawa et al., J. Org. Chem. 60: 925-930 (1995), claimed to have synthesized a decamer oligonucleotide containing a single methyl phosphotriester internucleotide linkage. However, NMR data supporting this claim was absent. Moreover, the method employed utilized costly and toxic palladium, which could contaminate the oligonucleotide product and render it unsuitable for therapeutic applications. In addition, the method was not shown to be able to introduce multiple methylphosphotriester linkages into the oligonucleotide.
There is therefore, a need for oligonucleotides containing methyl phosphotriester internucleotide linkages, as well as for new methods for synthesizing such oligonucleotides. Ideally, such oligonucleotides should be easy to synthesize and should be capable of containing numerous other beneficial modifications.